Characterization Complex Involved In Rothmund-Thomson

Rothmund Thomson综合征（RTS）是一种罕见的疾病，与基因组不稳定，易患癌症，皮肤和骨骼异常以及过早衰老的一些特征有关。该疾病是由RECQL 4突变引起的，RECQL 4是RecQ家族的推定解旋酶，其包括WRN和BLM，分别在Werner和Bloom综合征中有缺陷的蛋白质。为了了解这种疾病的机制，我们纯化了人RECQL 4复合物并鉴定了其相关成分。我们发现，大部分RECQL 4存在于HeLa细胞的细胞质提取物中，与大部分核BLM和WRN解旋酶相反。然而，在未转化的WI-38成纤维细胞中，RECQL 4被发现主要是核，并且以比转化的HeLa细胞中显著更低的总水平存在。来自HeLa细胞的RECQL 4作为与UBR 1和UBR 2的稳定复合物被分离。这些200 kD蛋白是N-末端规则途径的泛素连接酶，其底物包括具有不稳定N-末端残基的蛋白。这种蛋白水解途径的功能包括调节肽输入、染色体稳定性、减数分裂、细胞凋亡和心血管发育。虽然UBR 1和UBR 2的已知作用是介导其底物的多泛素化（和随后的降解），但UBR 1/2结合的RECQL 4在体内没有泛素化，并且在HeLa细胞中是一种长寿命的蛋白质。分离的RECQL 4-UBR 1/2复合物具有DNA刺激的ATP酶活性，但在基于DNA的解旋酶和易位酶测定中无活性，在该测定中BLM解旋酶是活性的。我们的数据表明，RECQL 4和泛素连接酶的N-末端规则途径可能在维持基因组稳定性中发挥作用。这些研究的第一份报告现在发表在《人类分子遗传学》上。

Rothmund Thomson Syndrome (RTS) is a rare disease associated with genome instability, predisposition to cancer, skin and skeletal abnormalities, and some features of premature aging. The disease is caused by mutation in RECQL4, a putative helicase of the RecQ family which includes WRN and BLM, proteins defective in Werner and Bloom syndromes, respectively. To understand the mechanism of this disease, we have purified a human RECQL4 complex and identified its associated components. We found that the bulk of RECQL4 was present in a cytoplasmic extract of HeLa cells, in contrast to the largely nuclear BLM and WRN helicases. However, in untransformed WI-38 fibroblasts, RECQL4 was found to be largely nuclear, and was present at significantly lower total levels than in transformed HeLa cells. RECQL4 from HeLa cells was isolated as a stable complex with UBR1 and UBR2. These 200 kD proteins are ubiquitin ligases of the N-end rule pathway, whose substrates include proteins with destabilizing N-terminal residues. The functions of this proteolytic pathway include the regulation of peptide import, chromosome stability, meiosis, apoptosis, and cardiovascular development. Although the known role of UBR1 and UBR2 is to mediate polyubiquitylation (and subsequent degradation) of their substrates, the UBR1/2-bound RECQL4 was not ubiquitylated in vivo, and was a long-lived protein in HeLa cells. The isolated RECQL4-UBR1/2 complex had a DNA-stimulated ATPase activity but was inactive in DNA-based assays for helicases and translocases, the assays in which the BLM helicase was active. Our data suggest that RECQL4 and ubiquitin ligases of the N-end rule pathway may play a role in maintaining genomic stability. The first report of these studies is now published in Human Molecular Genetics.